Myocardial injury after ischemia-reperfusion in mice deficient in Akt2 is associated with increased cardiac macrophage density

Akt2 protein kinase has been shown to promote cell migration and actin polymerization in several cell types, including macrophages. Because migrating macrophages constitute an important inflammatory response after myocardial ischemia, we determined cardiac macrophage expression after ischemia-reperfusion (I/R) injury and cryo-injury in mice lacking Akt2 (Akt2-KO). At 7 days post-I/R, Akt2-KO cardiac tissues showed an increase in immunohistochemical staining for macrophage markers (Galectin 3 and F4/80) compared with wild-type (WT) mice, indicating macrophage density was increased in the injured Akt2-KO myocardium. This change was time dependent because macrophage density was similar between WT and Akt2-KO myocardium at 3 days post-I/R, but by 7 and 14 days post-I/R, macrophage density was significantly increased in Akt2-KO myocardium. Concomitantly, infarct size was larger and cardiac function was reduced in Akt2-KO mice subjected to I/R. However, when cryo-infarction produced similar infarct sizes in the anterior wall in both WT and Akt2-KO mice, macrophage density remained higher in Akt2-KO mouse myocardium, suggesting Akt2 regulates myocardial macrophage density independent of infarct size. Consistently, bone marrow from Akt2-KO mice enhanced myocardial macrophage density in both C57/B6 WT and Akt2-KO recipient mice. Finally, reciprocal ex-vivo coculturing of macrophages and cardiac myocytes showed that activated Akt2-KO peritoneal macrophages had reduced mobility and adhesion when compared with WT littermate controls. Thus, although Akt-2 KO mice did not affect the initial inflammation response after injury and Akt2 deficiency has been shown to impair cell migration or motility in macrophages, our data suggested a novel mechanism in which increasing retention of Akt2-KO macrophages resulted in increasing cardiac Akt2-KO macrophage density in the myocardial space.     

Activation of non-canonical Wnt/JNK pathway by Wnt3a is associated with differentiation fate determination of human bone marrow stromal (mesenchymal) stem cells

The canonical Wnt signaling pathway can determine human bone marrow stromal (mesenchymal) stem cell (hMSC) differentiation fate into osteoblast or adipocyte lineages. However, its downstream targets in MSC are not well characterized. Thus, using DNA microarrays, we compared global gene expression patterns induced by Wnt3a treatment in two hMSC lines: hMSC-LRP5^T253 and hMSC-LRP5^T244 cells carrying known mutations of Wnt co-receptor LRP5 (T253I or T244M) that either enhances or represses canonical Wnt signaling, respectively. Wnt3a treatment of hMSC activated not only canonical Wnt signaling, but also the non-canonical Wnt/JNK pathway through upregulation of several non-canonical Wnt components e.g. naked cuticle 1 homolog (NKD1) and WNT11. Activation of the non-canonical Wnt/JNK pathway by anisomycin enhanced osteoblast differentiation whereas its inhibition by SP600125 enhanced adipocyte differentiation of hMSC. In conclusion, canonical and non-canonical Wnt signaling cooperate in determining MSC differentiation fate.     

C1q/Tumor Necrosis Factor-Related Protein 9 Protects against Acute Myocardial Injury through an Adiponectin Receptor I-AMPK-Dependent Mechanism

Obesity is a risk factor for cardiovascular disease. C1q/tumor necrosis factor-related protein 9 (CTRP9) is an adipokine that is downregulated by obesity. We investigated the role of CTRP9 in cardiac injury with loss-of-function genetic manipulations and defined the receptor-mediated signaling pathway downstream of this adipokine. CTRP9-knockout (CTRP9-KO) mice at the age of 12 weeks were indistinguishable from wild-type (WT) mice under basal conditions. CTRP9-KO mice had exacerbated contractile left ventricle dysfunction following intraperitoneal injection of lipopolysaccharide (LPS) compared to WT mice. Administration of LPS to CTRP9-KO mice also resulted in increased expression of proinflammatory cytokines and oxidative stress markers in the heart compared to WT mice. Likewise, CTRP9-KO mice showed increased myocardial infarct size and elevated expression of inflammatory mediators in ischemic heart following ischemia and reperfusion compared to WT mice. Treatment of cardiac myocytes with CTRP9 protein led to suppression of LPS-induced expression of proinflammatory genes, which was reversed by blockade of AMPK or ablation of adiponectin receptor I (AdipoR1). Systemic delivery of CTRP9 attenuated LPS-induced cardiac dysfunction in WT mice but not in muscle-specific transgenic mice expressing dominant-negative mutant form of AMPK or in AdipoR1-knockout mice. CTRP9 protects against acute cardiac damage in response to pathological stimuli by suppressing inflammatory reactions through AdipoR1/AMPK-dependent mechanisms.     

Sfrp1a and Sfrp5 function as positive regulators of Wnt and BMP signaling during early retinal development

Axial patterning of the developing eye is critically important for proper axonal pathfinding as well as for key morphogenetic events, such as closure of the optic fissure. The dorsal retina is initially specified by the actions of Bone Morphogenetic Protein (BMP) signaling, with such identity subsequently maintained by the Wnt-β catenin pathway. Using zebrafish as a model system, we demonstrate that Secreted frizzled-related protein 1a (Sfrp1a) and Sfrp5 work cooperatively to pattern the retina along the dorso-ventral axis. Sfrp1a/5 depleted embryos display a reduction in dorsal marker gene expression that is consistent with defects in BMP- and Wnt-dependent dorsal retina identity. In accord with this finding, we observe a marked reduction in transgenic reporters of BMP and Wnt signaling within the dorsal retina of Sfrp1a/5 depleted embryos. In contrast to studies in which canonical Wnt signaling is blocked, we note an increase in BMP ligand expression in Sfrp1a/5 depleted embryos, a phenotype similar to that seen in embryos with inhibited BMP signaling. Overexpression of a low dose of sfrp5 mRNA causes an increase in dorsal retina marker gene expression. We propose a model in which Sfrp proteins function as facilitators of both BMP and Wnt signaling within the dorsal retina.     

Secreted Frizzled-related Protein 1 (Sfrp1) Regulates the Progression of Renal Fibrosis in a Mouse Model of Obstructive Nephropathy

Renal fibrosis is responsible for progressive renal diseases that cause chronic renal failure. Sfrp1 (secreted Frizzled-related protein 1) is highly expressed in kidney, although little is known about connection between the protein and renal diseases. Here, we focused on Sfrp1 to investigate its roles in renal fibrosis using a mouse model of unilateral ureteral obstruction (UUO). In wild-type mice, the expression of Sfrp1 protein was markedly increased after UUO. The kidneys from Sfrp1 knock-out mice showed significant increase in expression of myofibrobast markers, α-smooth muscle actin (αSMA). Sfrp1 deficiency also increased protein levels of the fibroblast genes, vimentin, and decreased those of the epithelial genes, E-cadherin, indicated that enhanced epithelial-to-mesenchymal transition. There was no difference in the levels of canonical Wnt signaling; rather, the levels of phosphorylated c-Jun and JNK were more increased in the Sfrp1^−/− obstructed kidney. Moreover, the apoptotic cell population was significantly elevated in the obstructed kidneys from Sfrp1^−/− mice following UUO but was slightly increased in those from wild-type mice. These results indicate that Sfrp1 is required for inhibition of renal damage through the non-canonical Wnt/PCP pathway.     

SR-A deficiency reduces myocardial ischemia/reperfusion injury; involvement of increased microRNA-125b expression in macrophages

The macrophage scavenger receptor class A (SR-A) participates in the innate immune and inflammatory responses. This study examined the role of macrophage SR-A in myocardial ischemia/reperfusion (I/R) injury and hypoxia/reoxygenation (H/R)-induced cell damage. SR-A^?/? and WT mice were subjected to ischemia (45 min) followed by reperfusion for up to 7 days. SR-A^?/? mice showed smaller myocardial infarct size and better cardiac function than did WT I/R mice. SR-A deficiency attenuated I/R-induced myocardial apoptosis by preventing p53-mediated Bak-1 apoptotic signaling. The levels of microRNA-125b in SR-A^?/? heart were significantly greater than in WT myocardium. SR-A is predominantly expressed on macrophages. To investigate the role of SR-A macrophages in H/R-induced injury, we isolated peritoneal macrophages from SR-A deficient (SR-A^?/?) and wild type (WT) mice. Macrophages were subjected to hypoxia followed by reoxygenation. H/R markedly increased NF-κB binding activity as well as KC and MCP-1 production in WT macrophages but not in SR-A^?/? macrophages. H/R induced caspase-3/7 and -8 activities and cell death in WT macrophages, but not in SR-A^?/? macrophages. The levels of miR-125b in SR-A^?/? macrophages were significantly higher than in WT macrophages. Transfection of WT macrophages with miR-125b mimics attenuated H/R-induced caspase-3/7 and -8 activities and H/R-decreased viability, and prevented H/R-increased p-53, Bak-1 and Bax expression. The data suggest that SR-A deficiency attenuates myocardial I/R injury by targeting p53-mediated apoptotic signaling. SR-A^?/? macrophages contain high levels of miR-125b which may play a role in the protective effect of SR-A deficiency on myocardial I/R injury and H/R-induced cell damage.     

Brachy-syndactyly caused by loss of Sfrp2 function

Wnt signaling pathways are regulated both at the intracellular and extracellular levels. During embryogenesis, the in vivo effects of the secreted frizzled-related protein (Sfrp) family of Wnt inhibitors are poorly understood. Here, we show that inactivation of Sfrp2 results in subtle limb defects in mice with mesomelic shortening and consistent shortening of all autopodal elements that is clinically manifested as brachydactyly. In addition, there is soft-tissue syndactyly of the hindlimb. The brachydactyly is caused by decreased chondrocyte proliferation and delayed differentiation in distal limb chondrogenic elements. These data suggest that Sfrp2 can regulate both chondrogenesis and regression of interdigital mesenchyme in distal limb. Sfrp2 can also repress canonical Wnt signaling by Wnt1, Wnt9a, and Wnt4 in vitro. Sfrp2-/- and TOPGAL/Sfrp2-/- mice have a mild increase in beta-catenin and beta-galactosidase staining, respectively, in some phalangeal elements. This however does not exclude a potential concurrent effect on non-canonical Wnt signaling in the growth plate. In combination with what is known about BMP and Wnt signaling in human brachydactylies, our data establish a critical role for Sfrp2 in proper distal limb formation and suggest SFPR2 could be a novel candidate gene for human brachy-syndactyly defects.     

p21-activated kinase improves cardiac contractility during ischemia-reperfusion concomitant with changes in troponin-T and myosin light chain 2 phosphorylation

p21-activated kinase 1 (Pak1) is a serine/threonine kinase that activates protein phosphatase 2a, resulting in the dephosphorylation of cardiac proteins and increased myofilament Ca(2+) sensitivity. Emerging evidence indirectly indicates a role for Pak1 in ischemia-reperfusion (I/R), but direct evidence is lacking. We hypothesize that activation of the Pak1 signaling pathway is a cardioprotective mechanism that prevents or reverses the detrimental effects of ischemic injury by inducing posttranslational modifications in myofilament proteins that ultimately improve cardiac contractility following ischemic insult. In the present study, we subjected ex vivo hearts from wild-type (WT) and Pak1-knockout (KO) mice to 20 min of global cardiac ischemia followed by 30 min of reperfusion. In the absence of Pak1, there was an exacerbation of the increased end-diastolic pressure and reduced left ventricular developed pressure occurring after I/R injury. ProQ analysis revealed an increase in troponin-T phosphorylation at baseline in Pak1-KO hearts compared with WT. Significantly decreased myosin light chain 2 (MLC2) phosphorylation in Pak1-KO hearts compared with WT after I/R injury was confirmed by Western immunoblotting. These data indicate that Pak1-KO hearts have reduced recovery of myocardial performance after global I/R injury concomitant with changes in troponin-T and MLC2 phosphorylation. Finally, a protein-protein association between Pak1 and MLC2, and Pak1 and troponin-T, was determined by coimmunoprecipitation. Thus, results of our study provide a basis for targeting a novel pathway, including Pak1, in the therapies for patients with ischemic events.     

Secreted frizzled related protein 1 is a paracrine modulator of epithelial branching morphogenesis, proliferation, and secretory gene expression in the prostate

Previous in vitro studies identified secreted frizzled related protein 1 (SFRP1) as a candidate pro-proliferative signal during prostatic development and cancer progression. This study determined the in vivo roles of SFRP1 in the prostate using expression studies in mice and by creating loss- and gain-of-function mouse genetic models. Expression studies using an Sfrp1^lacZ knock-in allele showed that Sfrp1 is expressed in the developing mesenchyme/stroma of the prostate. Nevertheless, Sfrp1 null prostates exhibited multiple prostatic developmental defects in the epithelium including reduced branching morphogenesis, delayed proliferation, and increased expression of genes encoding prostate-specific secretory proteins. Interestingly, over-expression of SFRP1 in the adult prostates of transgenic mice yielded opposite effects including prolonged epithelial proliferation and decreased expression of genes encoding secretory proteins. These data demonstrated a previously unrecognized role for Sfrp1 as a stromal-to-epithelial paracrine modulator of epithelial growth, branching morphogenesis, and epithelial gene expression. To clarify the mechanism of SFRP1 action in the prostate, the response of WNT signaling pathways to SFRP1 was examined. Forced expression of SFRP1 in prostatic epithelial cells did not alter canonical WNT/β-catenin signaling or the activation of CamKII. However, forced expression of SFRP1 led to sustained activation of JNK, and inhibition of JNK activity blocked the SFRP1-induced proliferation of prostatic epithelial cells, suggesting that SFRP1 acts through the non-canonical WNT/JNK pathway in the prostate.     

Cardiac mTOR protects the heart against ischemia-reperfusion injury

Cardiac mammalian target of rapamycin (mTOR) is necessary and sufficient to prevent cardiac dysfunction in pathological hypertrophy. However, the role of cardiac mTOR in heart failure after ischemic injury remains undefined. To address this question, we used transgenic (Tg) mice with cardiac-specific overexpression of mTOR (mTOR-Tg mice) to study ischemia-reperfusion (I/R) injury in two animal models: 1) in vivo I/R injury with transient coronary artery ligation and 2) ex vivo I/R injury in Langendorff-perfused hearts with transient global ischemia. At 28 days after I/R, mortality was lower in mTOR-Tg mice than littermate control mice [wild-type (WT) mice]. Echocardiography and MRI demonstrated that global cardiac function in mTOR-Tg mice was preserved, whereas WT mice exhibited significant cardiac dysfunction. Masson's trichrome staining showed that 28 days after I/R, the area of interstitial fibrosis was smaller in mTOR-Tg mice compared with WT mice, suggesting that adverse left ventricular remodeling is inhibited in mTOR-Tg mice. In the ex vivo I/R model, mTOR-Tg hearts demonstrated improved functional recovery compared with WT hearts. Perfusion with Evans blue after ex vivo I/R yielded less staining in mTOR-Tg hearts than WT hearts, indicating that mTOR overexpression inhibited necrosis during I/R injury. Expression of proinflammatory cytokines, including IL-6 and TNF-α, in mTOR-Tg hearts was lower than in WT hearts. Consistent with this, IL-6 in the effluent post-I/R injury was lower in mTOR-Tg hearts than in WT hearts. These findings suggest that cardiac mTOR overexpression in the heart is sufficient to provide substantial cardioprotection against I/R injury and suppress the inflammatory response.     

Sfrp5 is not essential for axis formation in the mouse

Secreted frizzled related protein (Sfrp) genes encode extracellular factors that can modulate Wnt signaling. During early post-implantation mouse development Sfrp5 is expressed in the anterior visceral endoderm (AVE) and the ventral foregut endoderm. The AVE is important in anterior-posterior axis formation and the ventral foregut endoderm contributes to multiple gut tissues. Here to determine the essential role of Sfrp5 in early mouse development we generated Sfrp5-deficient mice by gene targeting. We report that Sfrp5-deficient mice are viable and fertile. To determine whether the absence of an axis phenotype might be due to genetic redundancy with Dkk1 in the AVE we generated Sfrp5;Dkk1 double mutant mice. AVE development and primitive streak formation appeared normal in Sfrp5(-/-);Dkk1(-/-) embryos. These results indicate that Sfrp5 is not essential for axis formation or foregut morphogenesis in the mouse and also imply that Sfrp5 and Dkk1 together are not essential for AVE development.     

Dickkopf-1 antagonizes Wnt signaling independent of beta-catenin in human mesothelioma

Dickkopf-1 (Dkk-1) is a secreted protein that acts as a potent inhibitor of the Wnt signal transduction pathway. It is thought that the antagonistic effect of Dkk-1 is specific to the canonical (Wnt/beta-catenin) pathway. In this study, we demonstrate that restoration of Dkk-1 expression suppresses cell growth and induces apoptotic cell death in beta-catenin-deficient mesothelioma cell lines H28 and MS-1. Furthermore, we found that a small-molecule inhibitor of JNK inhibited the apoptosis induced by Dkk-1 overexpression in these cells. Together, our data suggest that Dkk-1 may be able to antagonize Wnt signaling and exert its tumor suppressive effects through beta-catenin-independent non-canonical pathways (i.e., the Wnt/JNK pathway).     

Sfrp1 and Sfrp2 are not involved in Wnt/β-catenin signal silencing during lens induction but are required for maintenance of Wnt/β-catenin signaling in lens epithelial cells

During eye lens development, regulation of Wnt/β-catenin signaling is critical for two major processes: initially it must be silent in the lens placode for lens development to proceed, but subsequently it is required for maintenance of the lens epithelium. It is not known how these different phases of Wnt/β-catenin activity/inactivity are regulated. Secreted frizzled related protein-2 (Sfrp2), a putative Wnt-Fz antagonist, is expressed in lens placode and in lens epithelial cells and has been put forward as a candidate for regional Wnt/β-catenin pathway regulation. Here we show its closely-related isoform, Sfrp1, has a complimentary pattern of expression in the lens, being absent from the placode and epithelium but expressed in the fibers. As mice with single knockouts of Sfrp1 or Sfrp2 had no defects in lens formation, we examined lenses of Sfrp1 and Sfrp2 double knockout (DKO) mice and showed that they formed lens placode and subsequent lens structures. Consistent with this we did not observe ectopic TCF/Lef activity in lens placode of DKOs. This indicates that Sfrp1 and Sfrp2 individually, or together, do not constitute the putative negative regulator that blocks Wnt/β-catenin signaling during lens induction. In contrast, Sfrp1 and Sfrp2 appear to have a positive regulatory function because Wnt/β-catenin signaling in lens epithelial cells was reduced in Sfrp1 and Sfrp2 DKO mice. Lenses that formed in DKO mice were smaller than controls and exhibited a deficient epithelium. Thus Sfrps play a role in lens development, at least in part, by regulating aspects of Wnt/β-catenin signaling in lens epithelial cells.     

Wnt5a induces homodimerization and activation of Ror2 receptor tyrosine kinase

Wnts are secreted glycoproteins that control vital biological processes, including embryogenesis, organogenesis and tumorigenesis. Wnts are classified into several subfamilies depending on the signaling pathways they activate, with the canonical subfamily activating the Wnt/beta-catenin pathway and the non-canonical subfamily activating a variety of other pathways, including the Wnt/calcium signaling and the small GTPase/c-Jun NH2-terminal kinase pathway. Wnts bind to a membrane receptor Frizzled and a co-receptor, the low-density lipoprotein receptor related protein. More recently, both canonical and non-canonical Wnts were shown to bind the Ror2 receptor tyrosine kinase. Ror2 is an orphan receptor that plays crucial roles in skeletal morphogenesis and promotes osteoblast differentiation and bone formation. Here we examine the effects of a canonical Wnt3a and a non-canonical Wnt5a on the signaling of the Ror2 receptor. We demonstrate that even though both Wnt5a and Wnt3a bound Ror2, only Wnt5a induced Ror2 homo-dimerization and tyrosine phosphorylation in U2OS human osteoblastic cells. Furthermore, Wnt5a treatment also resulted in increased phosphorylation of the Ror2 substrate, 14-3-3beta scaffold protein, indicating that Wnt5a binding causes activation of the Ror2 signaling cascade. Functionally, Wnt5a recapitulated the Ror2 activation phenotype, enhancing bone formation in the mouse calvarial bone explant cultures and potentiating osteoblastic differentiation of human mesenchymal stem cells. The effect of Wnt5a on osteoblastic differentiation was largely abolished upon Ror2 down-regulation. Thus we show that Wnt5a activates the classical receptor tyrosine kinase signaling cascade through the Ror2 receptor in cells of osteoblastic origin.     

Disheveled mediated planar cell polarity signaling is required in the second heart field lineage for outflow tract morphogenesis

Disheveled (Dvl) is a key regulator of both the canonical Wnt and the planar cell polarity (PCP) pathway. Previous genetic studies in mice indicated that outflow tract (OFT) formation requires Dvl1 and 2, but it was unclear which pathway was involved and whether Dvl1/2-mediated signaling was required in the second heart field (SHF) or the cardiac neural crest (CNC) lineage, both of which are critical for OFT development. In this study, we used Dvl1/2 null mice and a set of Dvl2 BAC transgenes that function in a pathway-specific fashion to demonstrate that Dvl1/2-mediated PCP signaling is essential for OFT formation. Lineage-specific gene-ablation further indicated that Dvl1/2 function is dispensable in the CNC, but required in the SHF for OFT lengthening to promote cardiac looping. Mutating the core PCP gene Vangl2 and non-canonical Wnt gene Wnt5a recapitulated the OFT morphogenesis defects observed in Dvl1/2 mutants. Consistent with genetic interaction studies suggesting that Wnt5a signals through the PCP pathway, Dvl1/2 and Wnt5a mutants display aberrant cell packing and defective actin polymerization and filopodia formation specifically in SHF cells in the caudal splanchnic mesoderm (SpM), where Wnt5a and Dvl2 are co-expressed specifically. Our results reveal a critical role of PCP signaling in the SHF during early OFT lengthening and cardiac looping and suggest that a Wnt5a→ Dvl PCP signaling cascade may regulate actin polymerization and protrusive cell behavior in the caudal SpM to promote SHF deployment, OFT lengthening and cardiac looping.     

Zebrafish Naked1 and Naked2 antagonize both canonical and non-canonical Wnt signaling

Wnt signaling controls a wide range of developmental processes and its aberrant regulation can lead to disease. To better understand the regulation of this pathway, we identified zebrafish homologues of Naked Cuticle (Nkd), Nkd1 and Nkd2, which have previously been shown to inhibit canonical Wnt/beta-catenin signaling. Zebrafish nkd1 expression increases substantially after the mid-blastula transition in a pattern mirroring that of activated canonical Wnt/beta-catenin signaling, being expressed in both the ventrolateral blastoderm margin and also in the axial mesendoderm. In contrast, zebrafish nkd2 is maternally and ubiquitously expressed. Overexpression of Nkd1 or Nkd2a suppressed canonical Wnt/beta-catenin signaling at multiple stages of early zebrafish development and also exacerbated the cyclopia and axial mesendoderm convergence and extension (C&E) defect in the non-canonical Wnt/PCP mutant silberblick (slb/wnt11). Thus, Nkds are sufficient to antagonize both canonical and non-canonical Wnt signaling. Reducing Nkd function using antisense morpholino oligonucleotides resulted in increased expression of canonical Wnt/beta-catenin target genes. Finally, reducing Nkd1 function in slb mutants suppressed the axial mesendoderm C&E defect. These data indicate that zebrafish Nkd1 and Nkd2 function to limit both canonical and non-canonical Wnt signaling.     

Wnt5a Potentiates U46619-Induced platelet aggregation <Emphasis Type="Italic">via</Emphasis> the PI3K/Akt pathway

Platelet aggregation plays crucial roles in the formation of hemostatic plugs and thrombosis. Although it was recently shown that canonical Wnt signaling negatively regulates platelet aggregation, the role of non-canonical Wnt signaling remains unknown. Here, we observed that Wnt5a, one of the non-canonical Wnts, positively regulated platelet aggregation. Platelet aggregation was potentiated by the addition of Wnt5a to collagen-or U46619-induced rat platelet rich plasma (PRP). Treatment with Wnt5a to U46619-stimulated PRP resulted in an increase in the level of phosphorylated Akt, whereas phosphorylation of PKCδ and JNK1 was unaffected. In addition, inhibition of PI3K blocked the potentiating effect of Wnt5a. Taken together, these results suggest that Wnt5a potentiates U46619-induced platelet aggregation via the PI3K/Akt pathway.